Project Summary/Abstract Background: Radiation therapy (RT) is a core treatment modality that benefits patients with many types of cancer and can synergize with immune checkpoint blockade therapy. However, delivery of maximally effective doses of radiation to tumors is limited by collateral damage to normal tissues. We are developing a next- generation clinical RT platform called PHASER that will deliver ultra-rapid and precise radiation (FLASH) to decrease damage to normal tissues dramatically. Using a unique preclinical FLASH irradiator we developed for mice, our preliminary data show enhanced tumor control with FLASH vs. conventional dose rate irradiation as well as increased infiltration of immune cells into the tumor, suggestive of an immune mediated mechanism. Hypothesis and objective: We hypothesize that FLASH will demonstrate a superior therapeutic index by comparison to conventional dose rate RT for multiple cancers, based not only on its precision but also on the induction of more potent anti-tumor immunity. We will test this hypothesis in experimental models of cancer. Specific Aims and Study Design: Aim 1: Compare the anti-tumor potency, safety and immunological effects of FLASH vs conventional dose rate RT in primary tumors: We will evaluate different doses of FLASH and compare its effects with maximally tolerated doses of conventional dose rate RT in both syngeneic and patient derived xenograft mouse models. In addition to assessing tumor growth, we will analyze the effects of FLASH on the immune response, both locally and systemically through the use CyTOF and our SCAFFOLDS algorithms. This approach will reveal where and which immune cell subsets become activated in successfully treated animals. We will also assess the immunologic correlates of reduced toxicity from FLASH. Aim 2: Analyze the therapeutic effects of FLASH alone and in combination with immune checkpoint antibodies in metastatic disease. To assess our hypothesis that FLASH in combination with PD-1 blockade will exhibit synergistic anti-tumor effects due to an enhanced system-wide immune response, we will study the effects of FLASH, alone and in combination with anti-PD-1, on tumors outside the radiation field, and assess the immune response as in Aim 1. Aim 3: Identify the immune cellular and molecular basis of FLASH efficacy. We will test the hypothesis that efficacy is dependent on T cells as well as antigen presenting dendritic cells (DCs) by treating tumors in Rag-2 KO and BATF3 KO mice, respectively, and will elucidate the role for these cells by transferring T cells or DCs from successfully treated mice to naive mice challenged with tumor. The specific subsets required for efficacy are expected to be those shown to expand in multiple tissues in Aim 1. Lastly, we will explore the role of Type I interferon and its receptor on DCs in the efficacy of FLASH. Expected Results and Impact: These experiments are expected to demonstrate that FLASH in combination with checkpoint therapy promotes durable tumor regression of primary and metastatic tumors with little damage to normal tissues, thus setting stage for evaluating FLASH in clinical trials.